血管生成在创伤性颞下颌关节骨性强直形成中的作用

The pathological process and pathogenesis of traumatic temporomandibular joint (TMJ) ankylosis remain unclear. Clinical and experimental studies have shown that fibrous ankylosis and bony ankylosis are not the same pathological processes. Only the TMJ trauma seriously enough can lead to bony ankylosis, whereas the relative milder TMJ trauma causes fibrous ankylosis. However, how the injury severity of TMJ affects the tissue differentiation in the joint space, which ultimately resulting in different outcomes, is ill-defined. We noted that fibrous ankylosis showed avascular dense fibrous tissue in the joint space, whereas bony ankylosis demonstrated abundant angiogenesis associated with endochondral ossification. Taking the angiogenic-osteogenic coupling into account, for the first time, we hypothesized that angiogenesis affected the outcomes of TMJ trauma, and the increased neovascularization in the joint space after severe TMJ trauma would promote the development of bony ankylosis. Based on a reliable animal model, the studies intends to verify the hypothesis through three ways by MRA,Immunohistochemical staining, Real-time PCR, Westernblot etc.: ①comparing the angiogenesis and blood flow between the bony and fibrous ankylosis, ② exploring the temporal and spatial expression differences of key factors regulating angiogenesis between the bony and fibrous ankylosis, and ③ using the angiogenesis inhibitor to intervene the formation of bony ankylosis. These studies will demonstrate the key roles of angiogenesis in the development of traumatic TMJ bony ankylosis, and provide new ideas for the prevention and treatment of the condition.

创伤性颞下颌关节（TMJ）强直的发病过程及机制不清。临床与实验研究表明：纤维性和骨性强直并非同一病理学过程，足够严重的TMJ创伤方可导致骨性强直，相对轻微的TMJ创伤导致纤维性强直。然而，TMJ创伤程度如何影响关节间隙的组织分化并导致不同结局的机制尚不清楚。我们注意到纤维性强直表现为关节间隙无血管的纤维组织，而骨性强直表现为丰富的血管生成伴软骨内化骨。考虑到血管生成与骨形成紧密偶联，我们首次假设血管生成影响TMJ创伤后结局，严重的TMJ创伤导致关节间隙新生血管增加，促进骨性强直形成。本研究拟采用成熟的动物模型，通过磁共振成像、免疫组织化学染色、Real-time PCR、Westernblot等技术比较骨性及纤维性强直形成中：①血管生成及血流量的差异,②调节血管生成关键因子的时空表达差异。③采用血管生成抑制剂干预骨性强直。该研究将阐明血管生成在骨性强直形成中的关键作用，为其防治提供新思路。

TMJ创伤程度如何影响关节间隙的组织分化并导致不同结局的机制尚不清楚，我们推测关节间隙血管生成的差异影响了创伤后修复的结局。通过构建动物模型，我们发现，在其他创伤条件相同的情况下（即同时满足髁突矢状骨折+关节盘切除+关节窝表面纤维带剥离），髁突表面纤维带的创伤程度决定了髁突骨折的结局。纤维带完全去除为骨性强直，纤维带保留为骨关节炎，纤维带部分破裂为纤维性强直。该结果更新了目前关于关节强直发生条件的认识，为阐明关节强直发生的关键条件提供了实验依据。. 对7天、14天、28天、56天4个时点关节间隙组织块进行组织学及分子生物学检测，结果发现，创伤后早期特别是伤后第2周，骨性强直组血管生成（CD31的表达）显著高于纤维性强直组（P<0.05），而伤后第4周，骨性强直组血管生成（CD31的表达）显著低于纤维性强直组（P<0.05）。两组调节血管生成相关的基因MMP9、VEGFa, VEGFR2, Ang1, Ang2, Tie2的表达在多个时点存在差异。相对应的，调节血管内皮前体细胞趋化因子及粘附分子MCP1, SDF‐1及Hif‐1a的表达在多个时点存在差异。上述结果表明，差异性血管生成，特别是早期增强的血管生成在骨性强直形成过程中发挥重要作用。. 对4个时点关节间隙组织块进行原代细胞的分离培养，对第三代细胞进行了流式细胞仪鉴定，结果表明它们几乎不表达造血干细胞的表面标记物CD31和CD45，而高表达间充质干细胞表面标志物CD73、CD90和CD105。对第7天和第14天取材的细胞成功进行了成骨、成脂肪和成软骨诱导，表明它们为具有多向分化潜能的间充质干细胞。克隆增殖实验显示，纤维性强直关节间隙的间充质干细胞与骨强直关节间隙来源的间充质干细胞具有类似的增殖的潜能（58% Vs 61%，差异无统计学意义）和类似的成脂肪能力，但后者具有更强的成骨、成软骨能力。该结果表明关节间隙间充质细胞增强的成骨和成软骨潜能有助于骨性强直的形成。